Spinach hybrid SV4333VC and parents thereof

ABSTRACT

The invention provides seed and plants of spinach hybrid SV4333VC and the parent lines thereof. The invention thus relates to the plants, seeds and tissue cultures of spinach hybrid SV4333VC and the parent lines thereof, and to methods for producing a spinach plant produced by crossing such plants with themselves or with another spinach plant, such as a plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of such plants, including the leaf and gametes of such plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Appl. Ser. No. 62/062,093, filed Oct. 9, 2014, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, more specifically, to the development of spinach hybrid SV4333VC, and parent spinach lines SEBS011-1163F and SEB-66-0025D.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits in a single variety/hybrid. Such desirable traits may include any trait deemed beneficial by a grower and/or consumer, including greater yield, resistance to insects or pests, tolerance to environmental stress, better agronomic quality, higher nutritional value, growth rate and fruit properties.

Breeding techniques take advantage of a plant's method of pollination. There are two general methods of pollination: a plant self-pollinates if pollen from one flower is transferred to the same or another flower of the same plant or plant variety. A plant cross-pollinates if pollen comes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny, a homozygous plant. A cross between two such homozygous plants of different genotypes produces a uniform population of hybrid plants that are heterozygous for many gene loci. Conversely, a cross of two plants each heterozygous at a number of loci produces a population of hybrid plants that differ genetically and are not uniform. The resulting non-uniformity makes performance unpredictable.

The development of uniform varieties requires the development of homozygous inbred plants, the crossing of these inbred plants, and the evaluation of the crosses. Pedigree breeding and recurrent selection are examples of breeding methods that have been used to develop inbred plants from breeding populations. Those breeding methods combine the genetic backgrounds from two or more plants or various other broad-based sources into breeding pools from which new lines and hybrids derived therefrom are developed by selfing and selection of desired phenotypes. The new lines and hybrids are evaluated to determine which of those have commercial potential.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a spinach plant of the hybrid designated SV4333VC, the spinach line SEBS011-1163F, or SEB-66-0025D. Also provided are spinach plants having all the physiological and morphological characteristics of such a plant. Parts of these spinach plants are also provided, for example, including pollen, an ovule, scion, a rootstock, a fruit, and a cell of the plant.

In another aspect of the invention, a plant of spinach hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D comprising an added heritable trait is provided. The heritable trait may comprise a genetic locus that is, for example, a dominant or recessive allele. In one embodiment of the invention, a plant of spinach hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D is defined as comprising a single locus conversion. In specific embodiments of the invention, an added genetic locus confers one or more traits such as, for example, herbicide tolerance, insect resistance, disease resistance, and modified carbohydrate metabolism. In further embodiments, the trait may be conferred by a naturally occurring gene introduced into the genome of a line by backcrossing, a natural or induced mutation, or a transgene introduced through genetic transformation techniques into the plant or a progenitor of any previous generation thereof. When introduced through transformation, a genetic locus may comprise one or more genes integrated at a single chromosomal location.

The invention also concerns the seed of spinach hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D. The spinach seed of the invention may be provided as an essentially homogeneous population of spinach seed of spinach hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D. Essentially homogeneous populations of seed are generally free from substantial numbers of other seed. Therefore, seed of hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D may be defined as forming at least about 97% of the total seed, including at least about 98%, 99% or more of the seed. The seed population may be separately grown to provide an essentially homogeneous population of spinach plants designated SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D.

In yet another aspect of the invention, a tissue culture of regenerable cells of a spinach plant of hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D is provided. The tissue culture will preferably be capable of regenerating spinach plants capable of expressing all of the physiological and morphological characteristics of the starting plant, and of regenerating plants having substantially the same genotype as the starting plant. Examples of some of the physiological and morphological characteristics of the hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D include those traits set forth in the tables herein. The regenerable cells in such tissue cultures may be derived, for example, from embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistils, flowers, seed and stalks. Still further, the present invention provides spinach plants regenerated from a tissue culture of the invention, the plants having all the physiological and morphological characteristics of hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D.

In still yet another aspect of the invention, processes are provided for producing spinach seeds, plants and fruit, which processes generally comprise crossing a first parent spinach plant with a second parent spinach plant, wherein at least one of the first or second parent spinach plants is a plant of spinach line SEBS011-1163F or SEB-66-0025D. These processes may be further exemplified as processes for preparing hybrid spinach seed or plants, wherein a first spinach plant is crossed with a second spinach plant of a different, distinct genotype to provide a hybrid that has, as one of its parents, a plant of spinach line SEBS011-1163F or SEB-66-0025D. In these processes, crossing will result in the production of seed. The seed production occurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing” comprises planting seeds of a first and second parent spinach plant, often in proximity so that pollination will occur for example, mediated by insect vectors. Alternatively, pollen can be transferred manually. Where the plant is self-pollinated, pollination may occur without the need for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first and second parent spinach plants into plants that bear flowers. A third step may comprise preventing self-pollination of the plants, such as by emasculating the flowers (i.e., killing or removing the pollen).

A fourth step for a hybrid cross may comprise cross-pollination between the first and second parent spinach plants. Yet another step comprises harvesting the seeds from at least one of the parent spinach plants. The harvested seed can be grown to produce a spinach plant or hybrid spinach plant.

The present invention also provides the spinach seeds and plants produced by a process that comprises crossing a first parent spinach plant with a second parent spinach plant, wherein at least one of the first or second parent spinach plants is a plant of spinach hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D. In one embodiment of the invention, spinach seed and plants produced by the process are first generation (F₁) hybrid spinach seed and plants produced by crossing a plant in accordance with the invention with another, distinct plant. The present invention further contemplates plant parts of such an F₁ hybrid spinach plant, and methods of use thereof. Therefore, certain exemplary embodiments of the invention provide an F₁ hybrid spinach plant and seed thereof.

In still yet another aspect, the present invention provides a method of producing a plant derived from hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D, the method comprising the steps of: (a) preparing a progeny plant derived from hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D, wherein said preparing comprises crossing a plant of the hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D with a second plant; and (b) crossing the progeny plant with itself or a second plant to produce a seed of a progeny plant of a subsequent generation. In further embodiments, the method may additionally comprise: (c) growing a progeny plant of a subsequent generation from said seed of a progeny plant of a subsequent generation and crossing the progeny plant of a subsequent generation with itself or a second plant; and repeating the steps for an additional 3-10 generations to produce a plant derived from hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D. The plant derived from hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D may be an inbred line, and the aforementioned repeated crossing steps may be defined as comprising sufficient inbreeding to produce the inbred line. In the method, it may be desirable to select particular plants resulting from step (c) for continued crossing according to steps (b) and (c). By selecting plants having one or more desirable traits, a plant derived from hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D is obtained which possesses some of the desirable traits of the line/hybrid as well as potentially other selected traits.

In certain embodiments, the present invention provides a method of producing food or feed comprising: (a) obtaining a plant of spinach hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D, wherein the plant has been cultivated to maturity, and (b) collecting at least one spinach from the plant.

In still yet another aspect of the invention, the genetic complement of spinach hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D is provided. The phrase “genetic complement” is used to refer to the aggregate of nucleotide sequences, the expression of which sequences defines the phenotype of, in the present case, a spinach plant, or a cell or tissue of that plant. A genetic complement thus represents the genetic makeup of a cell, tissue or plant, and a hybrid genetic complement represents the genetic make up of a hybrid cell, tissue or plant. The invention thus provides spinach plant cells that have a genetic complement in accordance with the spinach plant cells disclosed herein, and seeds and plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles, and by the expression of phenotypic traits that are characteristic of the expression of the genetic complement, e.g., isozyme typing profiles. It is understood that hybrid SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D could be identified by any of the many well known techniques such as, for example, Simple Sequence Length Polymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858, specifically incorporated herein by reference in its entirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al., Science, 280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybrid genetic complements, as represented by spinach plant cells, tissues, plants, and seeds, formed by the combination of a haploid genetic complement of a spinach plant of the invention with a haploid genetic complement of a second spinach plant, preferably, another, distinct spinach plant. In another aspect, the present invention provides a spinach plant regenerated from a tissue culture that comprises a hybrid genetic complement of this invention.

Any embodiment discussed herein with respect to one aspect of the invention applies to other aspects of the invention as well, unless specifically noted.

The term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted otherwise. The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any plant that “comprises,” “has” or “includes” one or more traits is not limited to possessing only those one or more traits and covers other unlisted traits.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants, seeds and derivatives of spinach hybrid SV4333VC, spinach line SEBS011-1163F or SEB-66-0025D.

Spinach hybrid SV4333VC is resistant against Peronospora farinosa fsp. spinaciae races Pfs 1 to Pfs 12, 14 and 15. Hybrid SV4333VC can be characterized as an early bolting smooth hybrid that has spineless seeds. The hybrid is phenotypically distinct from all well-known material by a combination of Pfs 1 to 12, 14 and 15 resistances, early maturity and stemy leaves. The hybrid is believed to most closely resemble Seminis variety Solomon. The comparative characteristics that mostly distinguish to two hybrids include, but may not be limited to, resistances, maturity and stem length. The candidate variety SV4333VC has a different resistance pattern, darker leaves and more erected leaf habit compared to Bahamas.

Parent line SEBS011-1163F can be characterized as an early bolting line that has spineless seeds (smooth) and with a resistance to downy mildew (Peronospora farinosa fsp. spinaciae (Pfs) races Pfs 1 to Pfs 10. The line is believed to be most closely resemble Monsanto parental line SEBS011-1164F. The comparative characteristics that mostly distinguish to two lines include, but may not be limited to, maturity and stalk length. The candidate line SEBS011-1163F has a slower maturity and shorter stalk length compared to SEBS011-1164F.

The development of the line can be summarized as follows.

A. Origin and Breeding History of Spinach Hybrid SV4333VC

Hybrid SV4333VC was developed by crossing line SEBS011-1163F with line SEB-66-0025D. The parent lines are uniform and stable, as is a hybrid produced therefrom. A small percentage of variants can occur within commercially acceptable limits for almost any characteristic during the course of repeated multiplication. However no variants are expected.

B. Physiological and Morphological Characteristics of Spinach Hybrid SV4333VC, and Spinach Lines SEBS011-1163F and SEB-66-0025D

In accordance with one aspect of the present invention, there is provided a plant having the physiological and morphological characteristics of spinach hybrid SV4333VC and the parent lines thereof. A description of the physiological and morphological characteristics of such plants is presented in Tables 1-2.

TABLE 1 Physiological and Morphological Characteristics of Hybrid SV4333VC CHARACTERISTIC SV4333VC Solomon Ploidy diploid diploid Seedling Cotyledon length of cotyledon long long Width (mm) 8.2 mm 8.7 mm Length (mm) 94.8 mm 88.5 mm Tip pointed pointed Color medium green medium green Color Chart Name RHS RHS Color Chart Value 146b- 146b- Leaf (First Foliage Leaves) Shape ovate ovate Base V-shape V-shape Tip round-pointed round-pointed Margin flat flat Upper Surface Color medium green medium green (Giant Nobel) (Giant Nobel) Color Chart Name RHS RHS Color Chart Value 146b 146b Lower Surface Color lighter lighter (Compared with upper surface) Color Chart Name RHS RHS Color Chart Value 146c 146c Maturity Growth Rate medium (Long medium (Long Standing Standing Bloomsdale) Bloomsdale) Days from planting 42 40 to prime market stage Plant (Prime Market Stage) Habit semi-erect semi-erect (Long Standing (Long Standing Bloomsdale) Bloomsdale) Size large large (Giant Nobel) (Giant Noble) Spread (cm) 35.3 cm 31.8 cm Height (cm) 9.7 cm 11.3 cm Leaf (Prime Market Stage) Surface smooth smooth (Viroflay) (Viroflay) Anthocyanin coloration absent absent of petioles and veins Blade blistering weak weak Blade lobing weak medium Petiole: attitude semi-erect semi-erect Petiole: length long long (prime market stage) Blade attitude horizontal horizontal Leaf shape ovate ovate Blade shape (excluding medium ovate medium ovate basal lobes) Blade curving of margin flat flat Blade shape of apex obtuse obtuse Blade shape in flat flat longitudinal section Base lobed lobed Tip round-pointed round-pointed Margin slightly curved slightly curved Upper Surface Color medium green medium green Color Chart Name RHS RHS Color Chart Value 147A 147A Lower Surface Color lighter lighter (Compared with upper surface) Color Chart Name RHS RHS Color Chart Value 146A 146a Luster dull dull Blade size large medium Petiole color light green light green Petiole color RHS 144A 144A Petiole red pigmentation absent absent cm petiole length to the 11.6 cm 11.0 cm blade mm petiole diameter 7.3 mm 6.9 mm Petiole diameter medium medium Seed Stalk Development Start of Bolting (10% of late late plants) Height of Stalk (cm) 90.5 cm 83.6 cm Leaves on stalk of female many many plant Leaves on stalk of male many many plant Plants that are Female 36-65% 36-65% Proportion of female medium medium plants Plants that are Male 36-65% 36-65% Proportion of male plants medium medium Plants that are  0-10%  0-10% Monoecious Proportion of monoecious absent or absent or plants very low very low Seed Surface smooth smooth Spines (harvested seed) absent absent [Resistoflay] [Resistoflay] These are typical values. Values may vary due to environment. Other values that are substantially equivalent are also within the scone of the invention.

TABLE 2 Physiological and Morphological Characteristics of Line SEBS011-1163F CHARACTERISTIC SEBS011-1163F SEBS011-1164F Ploidy diploid diploid Seedling Cotyledon length of cotyledon long (Breedblad long (Breedblad Scherpzaad, Scherpzaad, Resistoflay) Resistoflay) Width (mm) 6.4 mm 8.1 mm Length (mm) 65.3 mm 111.9 mm Tip pointed pointed Color medium green medium green Color Chart Name RHS RHS Color Chart Value 146b 146b Leaf (First Foliage Leaves) Shape ovate ovate Base straight straight Tip round-pointed round-pointed Margin flat flat Upper Surface Color medium green medium green (Giant Nobel) (Giant Nobel) Color Chart Name RHS RHS Color Chart Value 146b 146b Lower Surface Color lighter lighter (Compared with upper surface) Color Chart Name RHS RHS Color Chart Value 146C 146C Maturity Growth Rate fast fast (Dixie Market) (Dixie Market) Days from planting to 36 38 prime market stage Plant (Prime Market Stage) Habit semi-erect semi-erect (Long Standing (Long Standing Bloomsdale) Bloomsdale) Size large large (Giant Nobel) (Giant Noble) Spread (cm) 42.5 cm 38.6 cm Height (cm) 14.7 cm 13.3 cm Leaf (Prime Market Stage) Surface smooth smooth (Viroflay) (Viroflay) Blade intensity of medium medium green color (Butterflay, (Butterflay, Monnopa) Monnopa) Petiole: attitude semi-erect semi-erect (Monnopa, (Monnopa, Parrot) Parrot) Petiole: length (prime medium (TG = market stage) Butterflay, Giraffe) Blade attitude semi-pendulous Semi-pendulous (Giraffe, (Giraffe, Medania) Medania) Blade shape (excluding broad ovate broad elliptic basal lobes) Blade curving of margin flat flat (Resistoflay) (Resistoflay) Blade shape of apex obtuse obtuse (Resistoflay) (Resistoflay) Blade shape in convex (Grappa, convex (Grappa, longitudinal section Lazio) Lazio) Base straight straight Tip round-pointed round-pointed Margin flat flat Upper Surface Color medium green medium green (Giant Nobel) (Giant Nobel) Color Chart Name RHS RHS Color Chart Value 146A 146B Lower Surface Color lighter lighter (Compared with upper surface) Color Chart Name RHS RHS Color Chart Value 146B 146C Luster dull dull Blade size large large (Giant Nobel) (Giant Nobel) Petiole color light green light green Petiole color RHS 143B 143B Petiole red pigmentation absent absent cm petiole length to the 10.1 cm 8.9 cm blade mm petiole diameter 7.8 mm 8.1 mm Petiole diameter medium medium Seed Stalk Development Start of Bolting (10% of early early plants) (Dixie Market) (Dixie Market) Height of Stalk (cm) 92.3 cm 82.0 cm Leaves on stalk of female many many plant Leaves on stalk of male many many plant Plants that are Female 0-10% (Monnopa) 0-10% (Monnopa) Plants that are Male 0-10% (Monnopa, 0-10% (Monnopa, Parrot) Parrot) Plants that are 91-100% 91-100% Monoecious (Monnopa) (Monnopa) Seed Surface smooth smooth Spines (harvested seed) absent absent [Resistoflay] [Resistoflay] These are typical values. Values may vary due to environment. Other values that are substantially equivalent are also within the scope of the invention.

C. Breeding Spinach Plants

One aspect of the current invention concerns methods for producing seed of spinach hybrid SV4333VC involving crossing spinach lines SEBS011-1163F and SEB-66-0025D. Alternatively, in other embodiments of the invention, hybrid SV4333VC, line SEBS011-1163F or line SEB-66-0025D may be crossed with itself or with any second plant. Such methods can be used for propagation of hybrid SV4333VC and/or the spinach lines SEBS011-1163F and SEB-66-0025D, or can be used to produce plants that are derived from hybrid SV4333VC and/or the spinach lines SEBS011-1163F and SEB-66-0025D. Plants derived from hybrid SV4333VC and/or the spinach lines SEBS011-1163F and SEB-66-0025D may be used, in certain embodiments, for the development of new spinach varieties.

The development of new varieties using one or more starting varieties is well known in the art. In accordance with the invention, novel varieties may be created by crossing hybrid SV4333VC followed by multiple generations of breeding according to such well known methods. New varieties may be created by crossing with any second plant. In selecting such a second plant to cross for the purpose of developing novel lines, it may be desired to choose those plants which either themselves exhibit one or more selected desirable characteristics or which exhibit the desired characteristic(s) when in hybrid combination. Once initial crosses have been made, inbreeding and selection take place to produce new varieties. For development of a uniform line, often five or more generations of selfing and selection are involved.

Uniform lines of new varieties may also be developed by way of double-haploids. This technique allows the creation of true breeding lines without the need for multiple generations of selfing and selection. In this manner true breeding lines can be produced in as little as one generation. Haploid embryos may be produced from microspores, pollen, anther cultures, or ovary cultures. The haploid embryos may then be doubled autonomously, or by chemical treatments (e.g. colchicine treatment). Alternatively, haploid embryos may be grown into haploid plants and treated to induce chromosome doubling. In either case, fertile homozygous plants are obtained. In accordance with the invention, any of such techniques may be used in connection with a plant of the invention and progeny thereof to achieve a homozygous line.

Backcrossing can also be used to improve an inbred plant. Backcrossing transfers a specific desirable trait from one inbred or non-inbred source to an inbred that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate locus or loci for the trait in question. The progeny of this cross are then mated back to the superior recurrent parent (A) followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny have the characteristic being transferred, but are like the superior parent for most or almost all other loci. The last backcross generation would be selfed to give pure breeding progeny for the trait being transferred.

The plants of the present invention are particularly well suited for the development of new lines based on the elite nature of the genetic background of the plants. In selecting a second plant to cross with SV4333VC and/or spinach lines SEBS011-1163F and SEB-66-0025D for the purpose of developing novel spinach lines, it will typically be preferred to choose those plants which either themselves exhibit one or more selected desirable characteristics or which exhibit the desired characteristic(s) when in hybrid combination. Examples of desirable traits may include, in specific embodiments, high seed yield, high seed germination, seedling vigor, high fruit yield, disease tolerance or resistance, and adaptability for soil and climate conditions. Consumer-driven traits, such as a fruit shape, color, texture, and taste are other examples of traits that may be incorporated into new lines of spinach plants developed by this invention.

D. Performance Characteristics

As described above, hybrid SV4333VC exhibits desirable traits, as conferred by spinach lines SEBS011-1163F and SEB-66-0025D. The performance characteristics of hybrid SV4333VC and spinach lines SEBS011-1163F and SEB-66-0025D were the subject of an objective analysis of the performance traits relative to other varieties. The results of the analysis are presented below.

TABLE 3 comparison SV4333VC with Solomon. Resistances Maturity Stem length SV4333VC Pfs 1-12, 14, 15 Earliest Longest Solomon Pfs 1-9, 11-15 latest shortest Location: Wageningen, the Netherlands 2012 to 2013.

TABLE 4 comparison SEBS011-1163F with SEBS011-1164F maturity Seed stalk length SEBS011-1163F slowest Longest SEBS011-1164F fastest shortest Location: Wageningen, the Netherlands.

E. Further Embodiments of the Invention

In certain aspects of the invention, plants described herein are provided modified to include at least a first desired heritable trait. Such plants may, in one embodiment, be developed by a plant breeding technique called backcrossing, wherein essentially all of the morphological and physiological characteristics of a variety are recovered in addition to a genetic locus transferred into the plant via the backcrossing technique. The term single locus converted plant as used herein refers to those spinach plants which are developed by a plant breeding technique called backcrossing, wherein essentially all of the morphological and physiological characteristics of a variety are recovered in addition to the single locus transferred into the variety via the backcrossing technique. By essentially all of the morphological and physiological characteristics, it is meant that the characteristics of a plant are recovered that are otherwise present when compared in the same environment, other than an occasional variant trait that might arise during backcrossing or direct introduction of a transgene.

Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the present variety. The parental spinach plant which contributes the locus for the desired characteristic is termed the nonrecurrent or donor parent. This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental spinach plant to which the locus or loci from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol.

In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (nonrecurrent parent) that carries the single locus of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a spinach plant is obtained wherein essentially all of the morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred locus from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original variety. To accomplish this, a single locus of the recurrent variety is modified or substituted with the desired locus from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological constitution of the original variety. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross; one of the major purposes is to add some commercially desirable trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered and the genetic distance between the recurrent and nonrecurrent parents. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele, or an additive allele (between recessive and dominant), may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

In one embodiment, progeny spinach plants of a backcross in which a plant described herein is the recurrent parent comprise (i) the desired trait from the non-recurrent parent and (ii) all of the physiological and morphological characteristics of spinach the recurrent parent as determined at the 5% significance level when grown in the same environmental conditions.

New varieties can also be developed from more than two parents. The technique, known as modified backcrossing, uses different recurrent parents during the backcrossing. Modified backcrossing may be used to replace the original recurrent parent with a variety having certain more desirable characteristics or multiple parents may be used to obtain different desirable characteristics from each.

With the development of molecular markers associated with particular traits, it is possible to add additional traits into an established germ line, such as represented here, with the end result being substantially the same base germplasm with the addition of a new trait or traits. Molecular breeding, as described in Moose and Mumm, 2008 (Plant Physiology, 147: 969-977), for example, and elsewhere, provides a mechanism for integrating single or multiple traits or QTL into an elite line. This molecular breeding-facilitated movement of a trait or traits into an elite line may encompass incorporation of a particular genomic fragment associated with a particular trait of interest into the elite line by the mechanism of identification of the integrated genomic fragment with the use of flanking or associated marker assays. In the embodiment represented here, one, two, three or four genomic loci, for example, may be integrated into an elite line via this methodology. When this elite line containing the additional loci is further crossed with another parental elite line to produce hybrid offspring, it is possible to then incorporate at least eight separate additional loci into the hybrid. These additional loci may confer, for example, such traits as a disease resistance or a fruit quality trait. In one embodiment, each locus may confer a separate trait. In another embodiment, loci may need to be homozygous and exist in each parent line to confer a trait in the hybrid. In yet another embodiment, multiple loci may be combined to confer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularly selected for in the development of a new inbred but that can be improved by backcrossing techniques. Single locus traits may or may not be transgenic; examples of these traits include, but are not limited to, herbicide resistance, resistance to bacterial, fungal, or viral disease, insect resistance, modified fatty acid or carbohydrate metabolism, and altered nutritional quality. These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as a dominant trait. For this selection process, the progeny of the initial cross are assayed for viral resistance and/or the presence of the corresponding gene prior to the backcrossing. Selection eliminates any plants that do not have the desired gene and resistance trait, and only those plants that have the trait are used in the subsequent backcross. This process is then repeated for all additional backcross generations.

Selection of spinach plants for breeding is not necessarily dependent on the phenotype of a plant and instead can be based on genetic investigations. For example, one can utilize a suitable genetic marker which is closely genetically linked to a trait of interest. One of these markers can be used to identify the presence or absence of a trait in the offspring of a particular cross, and can be used in selection of progeny for continued breeding. This technique is commonly referred to as marker assisted selection. Any other type of genetic marker or other assay which is able to identify the relative presence or absence of a trait of interest in a plant can also be useful for breeding purposes. Procedures for marker assisted selection are well known in the art. Such methods will be of particular utility in the case of recessive traits and variable phenotypes, or where conventional assays may be more expensive, time consuming or otherwise disadvantageous. Types of genetic markers which could be used in accordance with the invention include, but are not necessarily limited to, Simple Sequence Length Polymorphisms (SSLPs) (Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990), Randomly Amplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858, specifically incorporated herein by reference in its entirety), and Single Nucleotide Polymorphisms (SNPs) (Wang et al., Science, 280:1077-1082, 1998).

F. Plants Derived by Genetic Engineering

Many useful traits that can be introduced by backcrossing, as well as directly into a plant, are those which are introduced by genetic transformation techniques. Genetic transformation may therefore be used to insert a selected transgene into a plant of the invention or may, alternatively, be used for the preparation of transgenes which can be introduced by backcrossing. Methods for the transformation of plants that are well known to those of skill in the art and applicable to many crop species include, but are not limited to, electroporation, microprojectile bombardment, Agrobacterium-mediated transformation and direct DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wound tissues in a controlled manner.

An efficient method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a surface covered with target cells. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system for introducing gene loci into plant cells. An advantage of the technique is that DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations (Klee et al., Bio-Technology, 3(7):637-642, 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes. Additionally, Agrobacterium containing both armed and disarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene locus transfer. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (Fraley et al., Bio/Technology, 3:629-635, 1985; U.S. Pat. No. 5,563,055).

Transformation of plant protoplasts also can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al., Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature, 312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986; Marcotte et al., Nature, 335:454, 1988). Transformation of plants and expression of foreign genetic elements is exemplified in Choi et al. (Plant Cell Rep., 13: 344-348, 1994), and Ellul et al. (Theor. Appl. Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for any gene of interest including but not limited to selectable markers, scoreable markers, genes for pest tolerance, disease resistance, nutritional enhancements and any other gene of agronomic interest. Examples of constitutive promoters useful for plant gene expression include, but are not limited to, the cauliflower mosaic virus (CaMV) P-35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al., Nature, 313:810, 1985), including in monocots (see, e.g., Dekeyser et al., Plant Cell, 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); a tandemly duplicated version of the CaMV 35S promoter, the enhanced 35S promoter (P-e35S); 1 the nopaline synthase promoter (An et al., Plant Physiol., 88:547, 1988); the octopine synthase promoter (Fromm et al., Plant Cell, 1:977, 1989); and the figwort mosaic virus (P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter (P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem; the cauliflower mosaic virus 19S promoter; a sugarcane bacilliform virus promoter; a commelina yellow mottle virus promoter; and other plant DNA virus promoters known to express in plant cells.

A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals can also be used for expression of an operably linked gene in plant cells, including promoters regulated by (1) heat (Callis et al., Plant Physiol., 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter, Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a/b-binding protein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones, such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4) wounding (e.g., wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5) chemicals such as methyl jasmonate, salicylic acid, or Safener. It may also be advantageous to employ organ-specific promoters (e.g., Roshal et al., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988; Bustos et al., Plant Cell, 1:839, 1989).

Exemplary nucleic acids which may be introduced to plants of this invention include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. However, the term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and could potentially be introduced into a spinach plant according to the invention. Non-limiting examples of particular genes and corresponding phenotypes one may choose to introduce into a spinach plant include one or more genes for insect tolerance, such as a Bacillus thuringiensis (B.t.) gene, pest tolerance such as genes for fungal disease control, herbicide tolerance such as genes conferring glyphosate tolerance, and genes for quality improvements such as yield, nutritional enhancements, environmental or stress tolerances, or any desirable changes in plant physiology, growth, development, morphology or plant product(s). For example, structural genes would include any gene that confers insect tolerance including but not limited to a Bacillus insect control protein gene as described in WO 99/31248, herein incorporated by reference in its entirety, U.S. Pat. No. 5,689,052, herein incorporated by reference in its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, herein incorporated by reference in their entirety. In another embodiment, the structural gene can confer tolerance to the herbicide glyphosate as conferred by genes including, but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, herein incorporated by reference in its entirety, or glyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes by encoding a non-translatable RNA molecule that causes the targeted inhibition of expression of an endogenous gene, for example via antisense- or cosuppression-mediated mechanisms (see, for example, Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product (see for example, Gibson and Shillito, Mol. Biotech., 7:125, 1997). Thus, any gene which produces a protein or mRNA which expresses a phenotype or morphology change of interest is useful for the practice of the present invention.

G. Definitions

In the description and tables herein, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a gene locus, all of which alleles relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybrid progeny, for example a first generation hybrid (F₁), back to one of the parents of the hybrid progeny. Backcrossing can be used to introduce one or more single locus conversions from one genetic background into another.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes from different plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation of the organs with a cytoplasmic or nuclear genetic factor or a chemical agent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenic plants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets of chromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend to segregate together more often than expected by chance if their transmission was independent.

Marker: A readily detectable phenotype, preferably inherited in codominant fashion (both alleles at a locus in a diploid heterozygote are readily detectable), with no environmental variance component, i.e., heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, which characteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer to genetic loci that control to some degree numerically representable traits that are usually continuously distributed.

Resistance: As used herein, the terms “resistance” and “tolerance” are used interchangeably to describe plants that show no symptoms to a specified biotic pest, pathogen, abiotic influence or environmental condition. These terms are also used to describe plants showing some symptoms but that are still able to produce marketable product with an acceptable yield. Some plants that are referred to as resistant or tolerant are only so in the sense that they may still produce a crop, even though the plants are stunted and the yield is reduced.

Regeneration: The development of a plant from tissue culture.

Royal Horticultural Society (RHS) color chart value: The RHS color chart is a standardized reference which allows accurate identification of any color. A color's designation on the chart describes its hue, brightness and saturation. A color is precisely named by the RHS color chart by identifying the group name, sheet number and letter, e.g., Yellow-Orange Group 19A or Red Group 41B.

Self-pollination: The transfer of pollen from the anther to the stigma of the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed by a plant breeding technique called backcrossing, wherein essentially all of the morphological and physiological characteristics of a spinach variety are recovered in addition to the characteristics of the single locus transferred into the variety via the backcrossing technique and/or by genetic transformation.

Substantially Equivalent: A characteristic that, when compared, does not show a statistically significant difference (e.g., p=0.05) from the mean.

Tissue Culture: A composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant.

Transgene: A genetic locus comprising a sequence which has been introduced into the genome of a spinach plant by transformation.

H. Deposit Information

A deposit of spinach hybrid SV4333VC and inbred parent line SEBS011-1163F, disclosed above and recited in the claims, has been made with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The date of deposit was Oct. 17, 2014. The accession number for those deposited seeds of spinach hybrid SV4333VC and inbred parent line SEBS011-1163F are ATCC Accession No. PTA-121665 and ATCC Accession No. PTA-121666, respectively. Upon issuance of a patent, all restrictions upon the deposits will be removed, and the deposits are intended to meet all of the requirements of 37 C.F.R. §1.801-1.809. The deposits will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims.

All references cited herein are hereby expressly incorporated herein by reference. 

What is claimed is:
 1. A spinach plant comprising at least a first set of the chromosomes of spinach line SEBS011-1163F, a sample of seed of said line having been deposited under ATCC Accession Number PTA-121666.
 2. A seed comprising at least a first set of the chromosomes of spinach line SEBS011-1163F, a sample of seed of said line having been deposited under ATCC Accession Number PTA-121666.
 3. The plant of claim 1, which is an inbred.
 4. The plant of claim 1, which is a hybrid.
 5. The seed of claim 2, which is an inbred.
 6. The seed of claim 2, which is a hybrid.
 7. The plant of claim 4, wherein the hybrid plant is spinach hybrid SV4333VC, a sample of seed of said hybrid SV4333VC having been deposited under ATCC Accession Number PTA-121665.
 8. The seed of claim 6, wherein the seed is a seed of spinach hybrid SV4333VC, a sample of seed of said hybrid SV4333VC having been deposited under ATCC Accession Number PTA-121665.
 9. A plant part of the plant of claim
 1. 10. The plant part of claim 9, wherein the plant part is a leaf, an ovule, pollen, a fruit, or a cell.
 11. A spinach plant having all the physiological and morphological characteristics of the spinach plant of claim
 7. 12. A tissue culture of regenerable cells of the plant of claim
 1. 13. The tissue culture according to claim 12, comprising cells or protoplasts from a plant part selected from the group consisting of embryos, meristems, cotyledons, pollen, leaves, anthers, roots, root tips, pistil, flower, seed and stalks.
 14. A spinach plant regenerated from the tissue culture of claim 12 wherein said plant comprises all of the morphological and physiological characteristics of the spinach plant comprising at least a first set of the chromosomes of spinach line SEBS011-1163F, a sample of seed of said line having been deposited under ATCC Accession Number PTA-121666.
 15. A method of vegetatively propagating the spinach plant of claim 1 comprising the steps of: (a) collecting tissue capable of being propagated from said spinach plant; (b) cultivating said tissue to obtain proliferated shoots; and (c) rooting said proliferated shoots to obtain a rooted plantlet.
 16. The method of claim 15, further comprising growing at least a first spinach plant from said rooted plantlet.
 17. A method of introducing one desired trait into spinach line SEBS011-1163F, said method comprising: (a) crossing a plant of line SEBS011-1163F with a second spinach plant that comprises the desired trait to produce F1 progeny, a sample of seed of said line having been deposited under ATCC Accession Number PTA-121666; (b) selecting an F1 progeny that comprises the desired trait; (c) backcrossing the selected F1 progeny with a plant of the SEBS011-1163F line to produce backcross progeny; (d) selecting backcross progeny comprising the desired trait; and (e) repeating steps (c) and (d) three or more times to produce selected fourth or higher backcross progeny that comprise the desired trait and otherwise comprise of the morphological and physiological characteristics of line SEBS011-1163F.
 18. A spinach plant produced by the method of claim
 17. 19. A method of producing a spinach plant comprising an added trait, the method comprising introducing a transgene conferring the trait into a plant of spinach hybrid SV4333VC, or spinach line SEBS011-1163F, a sample of seed of said hybrid and line having been deposited under ATCC Accession Number PTA-121665, and ATCC Accession Number PTA-121666, respectively.
 20. A plant produced by the method of claim
 19. 21. A spinach plant comprising at least a first set of the chromosomes of spinach line SEBS011-1163F, a sample of seed of said line having been deposited under ATCC Accession Number PTA-121666, further comprising a transgene.
 22. The plant of claim 21, wherein said transgene confers a trait selected from the group consisting of male sterility, herbicide tolerance, insect resistance, pest resistance, disease resistance, modified fatty acid metabolism, environmental stress tolerance, modified carbohydrate metabolism and modified protein metabolism.
 23. The plant of claim 1, further comprising a single locus conversion.
 24. The plant of claim 23, wherein the single locus conversion confers a trait selected from the group consisting of male sterility, herbicide tolerance, insect resistance, pest resistance, disease resistance, modified fatty acid metabolism, environmental stress tolerance, modified carbohydrate metabolism and modified protein metabolism.
 25. A method for producing a seed of a spinach plant derived from at least one of spinach hybrid SV4333VC, or spinach line SEBS011-1163F comprising the steps of: (a) crossing the spinach plant of hybrid SV4333VC, or line SEBS011-1163F with itself or a second spinach plant; a sample of seed of said hybrid and line having been deposited under ATCC Accession Number PTA-121665, and ATCC Accession Number PTA-121666, respectively; and (b) allowing a seed of a hybrid SV4333VC-derived, or line SEBS011-1163F-derived spinach plant to form.
 26. The method of claim 25, further comprising the steps of: (c) selfing a plant grown from said hybrid SV4333VC-derived, or SEBS011-1163F-derived spinach seed to yield an additional hybrid SV4333VC-derived, or line SEBS011-1163F-derived spinach seed; (d) growing said additional hybrid SV4333VC-derived, or line SEBS011-1163F-derived spinach seed of step (c) to yield an additional hybrid SV4333VC-derived, or line SEBS011-1163F-derived spinach plant; and (e) repeating the crossing and growing steps of (c) and (d) to generate at least a first further hybrid SV4333VC-derived, or line SEBS011-1163F-derived spinach plant.
 27. The method of claim 25, wherein the second spinach plant is of an inbred spinach line.
 28. The method of claim 26, further comprising: (f) crossing the further hybrid SV4333VC-derived, or SEBS011-1163F-derived spinach plant with a second spinach plant to produce seed of a hybrid progeny plant.
 29. A plant part of the plant of claim
 7. 30. The plant part of claim 29, wherein the plant part is a leaf, an ovule, pollen, a fruit, or a cell.
 31. A method of producing a spinach seed comprising crossing the plant of claim 1 with itself or a second spinach plant and allowing seed to form.
 32. A method of producing a spinach leaf comprising: (a) obtaining the plant according to claim 1, wherein the plant has been cultivated to maturity; and (b) collecting the spinach leaf from the plant. 